This proposal details the development of a general phage display selection strategy for evolving protein-based inhibitors that contain an unnatural amino acid "warhead" for selectively targeting cell surface proteases known to be overexpressed in cancer. All known organisms encode the same 20 amino acids. However, considering the vast array of cofactors and posttranslational modifications that endow endogenous proteins with altered functionalities, it is possible that an expanded genetic code may provide an evolutionary advantage through the generation of proteins with novel functions or enhanced fitness. It has recently been demonstrated that modified tRNAs and aminoacyl-tRNA synthetase pairs are capable of incorporating unnatural amino acids into large scale antibody libraries for the purpose of carrying out functional selections. Herein is described an initial phage display system for the incorporation of unnatural amino acids into cyclic peptide inhibitors of a cell surface protease, MT-SP1 (matriptase). The specific aims are as follows: 1) Genetically encoding an unnatural amino acid warhead for targeting the MT-SP1 active site and 2) Phage display of cyclic peptides for directed evolution using an expanded genetic code. Ultimately this strategy should be general for the directed evolution of unnatural amino acids in the context of peptides or larger protein scaffolds for targeting any relevant biomolecule. PUBLIC HEALTH RELEVANCE: Proteases are involved in many diverse physiological processes. As a consequence, misregulation of protease function may correlate with a variety of pathological conditions ranging from cardiovascular disorders to cancer. Therefore, proteases are excellent targets for therapeutic intervention or diagnosis, and currently represent 5-10% of pharmaceutical targets. MT-SP1 was initially isolated from breast cancer cells and has been implicated in tissue remodeling associated with metastasis. Selective cyclic peptide affinity reagents should provide a valuable method for evaluating the overexpression of this protease, and may ultimately provide a basis for the discovery of novel diagnostics and therapeutics. PUBLIC HEALTH RELEVANCE: Proteases, such as MT-SP1, are involved in many diverse physiological processes. As a consequence, misregulation of function may correlate with a variety of pathological conditions ranging from cardiovascular disorders to cancer. Selective cyclic peptide affinity reagents targeted against MT-SP1 should provide a valuable method for evaluating the overexpression of this protease, and may ultimately provide a basis for the discovery of novel diagnostics and therapeutics.